This application is a 371 of PCT/US97/16890, filed Sep. 19, 1997.
The invention relates to materials which contain polysaccharide chains, particularly alginate or modified alginate chains. The polysaccharide, particularly alginate or modified alginate, chains may be included as side chains or auxiliary chains from a backbone polymer chain, which may also be a polysaccharide. Further, the polysaccharide chains may be crosslinked between side chains, auxiliary chains and/or backbone chains. These materials are advantageously modified by covalent bonding thereto of a biologically active molecule for cell adhesion or other cellular interaction. The materials are particularly useful to provide polymeric matrices for many applications, such as in tissue engineering applications for bone or soft tissue replacement. For example, the loss of bony tissue is a central feature of many aspects of clinical dentistry (e.g. periodontal disease, caries, osteotomy for repair of trauma) and matrices from the materials described herein can be useful for repair or replenishment of lost bony tissue. The materials are also useful for drug delivery applications when the biologically active molecule is attached by a degradeable bond.
Unmodified alginate, a polysaccharide, has been previously utilized as a tissue engineering matrix in cell encapsulation and transplantation studies. It provides a useful matrix because cells can be immobilized within alginate with little cell trauma and alginate/cell mixtures can be transplanted in a minimally invasive manner. However, cells exhibit little or no adhesion or interaction with unmodified alginate. One aspect of this invention is to provide a matrix which combines specific cell adhesion ligands in the matrix such that high control over cell-matrix interactions, due to cell adhesion and matrix interactions, is attained.
One embodiment of the invention is directed to polymers containing a polymer backbone to which is linked polysaccharide groups, particularly of alginates or modified alginates, which preferably are polymerized D-mannuronate and/or L-guluronate monomers. The polysaccharide, particularly alginate, groups are present as side chains on the polymer backbone which is intended to include side chains at the terminal end of the backbone, thus being a continuation of the main chain. The polymers provide synthetic modified polysaccharides and alginates exhibiting controllable properties depending upon the ultimate use thereof. Further, the invention is directed to processes for preparing such polymers and to the use of such polymers, for example, as cell transplantation matrices, preformed hydrogels for cell transplantation, non-degradable matrices for immunoisolated cell transplantation, vehicles for drug delivery, wound dressings and replacements for industrially applied alginates.
Another embodiment of the invention is directed to polysaccharides, particularly alginates, which are modified by being crosslinked. The alginates may further be modified by covalent bonding thereto of a biologically active molecule for cell adhesion or other cellular interaction. Crosslinking of the alginate can particularly provide aloinate materials with controlled mechanical properties and shape memory properties which greatly expand their range of use, for example, to tissue engineering applications where size and shape of the matrix is of importance. The modification of the crosslinked alginates with the biologically active molecules can provide a further three-dimensional environment which is particularly advantageous for cell adhesion, thus making such alginates further useful as cell transplantation matrices. Further, the invention is directed to processes for preparing such crosslinked alginates and to their use, for example, for forming materials for tissue engineering and/or having cell adhesion properties particularly for cell transplantation matrices, such as injectable cell transplantation solutions and preformed materials for cell transplantation.
Another embodiment of the invention is directed to modified alginates, such as alginate backbone (i.e. unmodified alginate) or the above described side chain alginates or crosslinked alginates, modified by covalent bonding thereto of a biologically active molecule for cell adhesion or other cellular interaction, which is particularly advantageous for maintenance, viability and directed expression of desirable patterns of gene expression. The modified alginate polymers provide a three-dimensional environment which is particularly advantageous for cell adhesion. Further, the invention is directed to processes for preparing such polymers and to the use of such polymers, for example, for forming gels or highly viscous liquids having cell adhesion properties particularly for cell transplantation matrices, such as injectable cell transplantation solutions and preformed hydrogels for cell transplantation.
Further aspects of the invention may be determined by one of ordinary skill in the art from the following description.
Organ or tissue failure remains a frequent, costly, and serious problem in health care despite advances in medical technology. Available treatments now include transplantation of organs from one individual to another, performing surgical reconstructing, use of mechanical devices (e.g., kidney dialyzer) and drug therapy. However, these treatments are not perfect solutions. Transplantation of organs is limited by the lack of organ donors, possible rejection and other complications. Mechanical devices cannot perform all functions of an organ, e.g., kidney dialysis can only help remove some metabolic wastes from the body. Likewise, drug levels comparable to the control systems of the body is difficult to achieve. This is partially due to difficulties in controlling the drug level in vivo. Financially, the cost of surgical procedures is very high. Advances in medical, biological and physical sciences have enabled the emergency of the field of tissue engineering. xe2x80x9cTissue engineeringxe2x80x9d is the application of the principles and methods of engineering and the life sciences toward the fundamental understanding of structure/function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain or improve function. It thus involves the development of methods to build biological substitutes as supplements or alternatives to whole organ or tissue transplantation . The use of living cells and/or extracellular matrix (ECM) components in the development of implantable parts or devices is an attractive approach to restore or to replace function. The advantage of this approach over whole organ/tissue transplantation is that only the cells of interest are implanted, and they potentially can be multiplied in vitro. Thus, a small biopsy can be grown into a large tissue mass and, potentially, could be used to treat many patients. The increased tissue supply may reduce the cost of the therapy because early intervention is possible during the disease, and this may prevent the long-term hospitalization which results as tissue failure progresses. The use of immunosuppression may also be avoided in some applications by using the patient""s own cells.
Alginate is a linear polysaccharide, isolated, for example, from brown sea algae, which forms a stable hydrogel in the presence of divalent cations (e.g., Ba++, Ca++) (Smidsrod et al (1990): Alginate as immobilization matrix for cells. TIBTECH, 8:71-78.) Alginate is currently being used for the in vitro culture of some cells types, as an injectable cell delivery matrix, for immunoisolation based therapies, and as an enzyme immobilization substrate (Atala et al., 1993: Injectable alginate seeded with chondrocytes as a potential treatment for vesicoureteral reflux. J. Urology, 150:745:747; Levesque et al., 1992: Maintenance of long-term secretory function by microencapsulated islets of Langerhans. Endocrinology, 130:644-650; Dominguez et al., 1988: Carbodiimide coupling of xcexc-galactosidase from Aspergillus oryzae to alginate. Enzyme Microb. Technol., 10:606-610; and Lee et al. 1993: Covalent Immobilization of Aminoacylase to Alginate for L-hphenylalanine production. J. Chem. Tech. Biotechnol, 58:65-70.). Alginate hydrogels are attractive for use with cells because of their mild gelling conditions, low diffusional barriers to cell nutrients, and low inflammatory and nontoxicity in vivo (Smidsrod, supra).
Alginates occur naturally as copolymers of D-mannuronate (M) and L-guluronate (G) and have different monomer compositions when isolated from different natural sources. The block length of monomer units, overall composition and molecular weight of the alginate influence its properties. For example, calcium alginates rich in G are stiff materials, (see Sutherland, I W (1991): Alginates. In Biomaterials.: Novel materials from biological sources.). It is theorized that gel formation is due primarily to the G-block, and that the M-block is essentially non-selective. In such arrangement, the calcium ions would be selectively bound between sequences of polyguluronate residues and held between diaxially linked L-guluronate residues which are in the 1C4 chair conformation. The calcium ions would thus be packed into the interstices between polyguluronate chains associated pairwise and this structure is named the xe2x80x9cegg-boxxe2x80x9d sequence. The ability to form a junction zone depends on the length of the G-blocks in different alginates (Sutherland, supra.). Other advantages of alginates include their wide availability, low diffusional barrier for all nutrients and relative biocompatibility (Smidsrod et al., Trends in Biotech, 8:71-78, 1990).
A limitation of alginate hydrogels used with a cellular component is the lack of inherent cell adhesion. Such is necessary for cell attachment and long term survival of most mammalian cell systems. While chrondrocytes and islets of Langerhans have been successfully transplanted using alginates, the absence of suitable cell adhesion by alginates practically limits their use to cartilage and islet cell applications. Most other cell types require attachment to an extracellular substrate to remain viable.
Previous attempts have been made to create a three-dimensional hydrogel environment incorporating cell adhesion ligands for cell attachment and survival. One system is a photopolymerizable polyacrylamide based hydrogel with an RGD peptide grafted onto the polymer backbone. This polymer undergoes photogelation in the presence of UV light, and may be polymerized as a polymer/cell hybrid (Moghaddam et al., 1993: Molecular design of three-dimensional artificial extracellular matrix: photosensitive polymers containing cell adhesive peptide. J. Polymer Science: Part A: Polymer Chem. 31:1589-1597.) Another is a polyacrylamide system, again with the RGD ligand covalently attached, which is catalytically polymerized prior to any biological interactions (Woerly et al. 1995: Intracerebral implantation of hydrogel-coupled adhesion peptides: tissue reaction. J. Neural Transplant. Plasticity, 5:245:255.). A disadvantage of such systems is that conversion of the polymers from a liquid to a solid, gel or highly viscous system requires conditions which are detrimental to cell viability, e.g., use of organic solvents and/or elevated temperatures.
Another major limitation of alginate hydrogels used in biotechnology applications is that their stability is dependent solely on calcium (or other divalent cation) binding, and this can present a limitation in the use of these materials (e.g., loss of calcium from gels leads to gel dissolution). In addition, alginate hydrogels have a limited range of physical properties due to the limited number of variables one can currently manipulate (i.e., alginate concentration, specific divalent cation used for gelling, and concentration of divalent cation). This limitation is especially evident when alginate is utilized as an injectable cell delivery vehicle in tissue engineering. It is not possible to obtain a pre-defined and desirable shape of the matrix following injection, and it is thus not possible to create a new tissue with a specific and desirable shape and size. This is especially important whenever the size and shape of the new tissue are critical to the function of the tissue, for example, in reconstruction of facial features such as nose or ears, or relining of joints.
An object of the present invention was to design improved synthetic analogues of alginates, to provide a process for preparing such polymers and to provide compositions and methods utilizing such polymers, particularly in tissue engineering applications. It is further useful according to the invention to provide alginate-containing materials in which the gel stability is related to an additional variable besides cation binding from the divalent cations. Thus, for example, the disadvantages of the previous systems can be avoided by providing an alginate which can be gelled or made highly viscous under mild conditions, i.e., in the presence of divalent metal cations such as Ca++or Ba++in aqueous systems, without requiring, for example, organic solvents and/or increased temperature.
In one embodiment, the invention provides polymers with side chains of polysaccharides in general which may not exhibit the gelling behavior of alginates, but which provide polysaccharides with controllable properties, such as degradation. These polymers may comprise a polymeric backbone section to which is covalently linked a polysaccharide side chain. Another embodiment provides a polymeric backbone section to which is bonded a side chain, preferably multiple side chains, of polymerized, optionally modified, D-mannuronate (M units) and/or L-guluronate (G units) monomers. The modified alginates preferably maintain the mild gelling behavior of conventional alginates, but do not have the disadvantages discussed above. The linkage between the polymeric backbone section and the side chain(s) may be provided by difunctional or multifunctional linker compounds, by groups incorporated within the polymeric backbone section reactive with the polysaccharide units and/or by groups on the polysaccharide units or derivatives thereof reactive with groups on the polymeric backbone section. The polymers may advantageously further comprise biologically active molecules bonded thereto, particularly preferably bonded through the carboxylic acid groups on M and/or G units. In a particularly preferred embodiment, the side chains are alginates, the biologically active molecules exhibit cell adhesion properties and the polymers are useful for cell transplantation.
An advantageous aspect of these materials is the ability to provide a polymer analogous to alginates, but with high controllability of the properties, particularly when used for cell transplantation purposes. The chemical structures, functionality and sizes of the different parts of the polymer, i.e., the backbone, linker, side chain and, optionally, biologically active molecule(s) can be provided so as to control many properties of the polymer in physiological systems, such as, for example, degradeability, biocompatibility, organ or tissue specificity and affinity, cell adhesion, cell growth and cell differentiation, manner and rate of removal from the system, solubility and viscosity.
As the polymeric backbone section there can be used any homo- or co-polymer which is compatible with the ultimate use and which has the appropriate functional groups such that it can be covalently linked directly or through a linker to the polysaccharide, particularly polymerized M and/or G units, or suitable modifications thereof. Any polymer meeting the above requirements is useful herein, and the selection of the specific polymer and acquisitions or preparation of such polymer would be conventionally practiced in the art. See The Biomedical Engineering Handbook, ed. Bronzino, Section 4, ed. Park. Preferred for such polymeric backbone section are, for example, poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), polypeptides, poly(amino acids), such as poly(lysine), poly(allylamines) (PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), polyesters, polyphosphazenes, pluronic polyols, polyoxamers, poly(uronic acids) and copolymers, including graft polymers thereof.
The polymeric backbone section may be selected to have a wide range of molecular weights, generally from as low as 100 up to ten million. However, by selection of the molecular weight and structure of the polymeric backbone section the occurrence and rate of degradeability of the polymer and the manner and rate of release from physiological systems of the polymer can be influenced. For instance, a high molecular weight non-degradable polymeric backbone section, for instance having a molecular weight above about 100,000, will in general provide a more stable polymer which may be useful in, for example, nondegradable matrices for immunoisolated cell transplantation. Alternatively, a polymeric backbone section having a molecular weight of less than about 30,000 to 50,000 or one in which the backbone itself is degradable can be cleared through the kidneys and by other normal metabolic routes. Polymers with a degradable polymeric backbone section include those with a backbone having hydrolyzable groups therein, such as polymers containing ester groups in the backbone, for example, aliphatic polyesters of the poly(a-hydroxy acids) including poly(glycolic acid) and poly(lactic acid). When the backbone is itself degradable, it need not be of low molecular weight to provide such degradeability. A particular example of a degradable polymer for the backbone is a graft polymer of PEO (polyethylene oxide) and acetyl-aspartate shown by the following equation, wherein the first equation shows formation of the degradable polymer backbone, and the second schematic shows the attachment of side chains thereto: 
A: DCC, HOBT, DMAP, DMF, B; X=OBT or OSu, NMM, DMF.
Copolymer content can be controlled by the block length of PEO and mixing in Ac-aspartate. 
The solubility, viscosity, biocompatibility, etc., of the polymeric backbone section also is a consideration as to its effect on the desired properties of the final polymer product.
In one embodiment the polymeric backbone section can be one which incorporates linkage sites for the polysaccharide side chains so that a separate linker group is not required. For example, poly(amino acids) having free amino groups may be used for this purpose.
When a linker group is used, such linker group may be selected from any divalent moieties which are compatible with the ultimate use of the polymer and which provide for covalent bonding between the polymeric backbone section and the polysaccharide side chain(s). Such linker groups are conventionally known in the art for such purpose, and can be linked to the backbone in a conventional manner. For linking of the linker or linkage site on the polymer to the polysaccharide, since the polysaccharide is generally bonded through a carboxylate group, chemistries useful for reacting with carboxylate groups are particularly useful in providing the linker or linkage site on the polymer; see Bronzino and Hermanson, cited below. The linker group may be selected to significantly affect the biodegradability of the polymer depending upon the extent of hydrolyzability of groups in the linker chain. Amino acid linkers and derivatives thereof are preferred due to the controllability of the degradation feature. For example, amino acid linker groups, such as glycine, will provide ester linkages which are readily hydrolyzable and, thus, facilitate degradation of the polymer in an aqueous environment, whereas, amino alcohols provide an ether linkage which is significantly less degradable. Amino aldehydes are also useful linker groups. The substituent groups on the amino acids will also affect the rate of degradeability of the linkage. The linker group may also be varied in chain length depending upon the desired properties. Linkages providing, for example, from 1 to 10 atoms between the backbone and side chain, are preferred, although longer linkage chains are possible. Further, the linker may be branched to provide multiple attachment sites for the side chains, for example, to provide a dendrite configuration such as shown in Example 5. The linker will be in the form of a residue of the linking compound without the group removed during bonding.
The side chains are polysaccharides, preferably optionally modified alginate units, which enable the preparation of a gel or highly viscous liquid in the presence of a divalent metal, e.g., Ca++or Ba++. Preferably they are comprised of polymerized D-mannuronate (M) and/or L-guluronate (G) monomers, but, also encompass modified such monomers. The side chains are particularly preferably comprised of oligomeric blocks of M units, G units or M and G units. The molecular weight of each side chain or the number of units and length of such side chains is again a function of the desired ultimate properties of the polymer and selectability of this aspect is an advantageous feature of the invention. Although there is no specific limitation, the molecular weight of the side chain may range from about 200 up to one million, and may contain, preferably 2 to 5,000 M and/or G units. As with the polymeric backbone section, higher molecular weight side chains, e.g. above about 100,000, are generally useful when more stable polymers are desired and lower molecular weight side chains, e.g., below about 30,000 to 50,000, are generally useful when biodegradable species capable of removal through the kidneys, or other normal functions, are desired.
The distribution of M and G units also provides a controllability feature of the invention with a higher ratio of G units generally providing a stiffer polymer which will hold its shape better. Side chains having a percentage of G units based on the total of M and G units of from 10 to 100% are particularly preferred. Increasing or decreasing the number of G units in the side chains will also allow for increasing or decreasing the rate of gelation of the polymer. Such may be of interest when the polymers are used in injectable solutions and the rate is controlled so that the solution will gel at the appropriate time after injection. The number of side chains provided on the polymeric backbone section also will affect the extent and rate of gelation and, thus, will vary depending on the ultimate use. In general, more side chains will result in a more rigid, compact polymer, and provide a more dense concentration of attached biologically active molecules, if present. The number of side chains is preferably from 1 to 100% of the reactive monomer units available on the backbone per polymer molecule. It is not necessary that every linker group or linkage site be provided with a side chain. For example, free linkers or linkage sites may be left to facilitate the solubility and/or compatibility of the polymer in its intended system. Additionally, free linkers or linkage sites may be provided to allow for the later addition of differently structured or proportioned alginate side chains or other side chains.
Furthermore, the whole side chain or individual M and/or G units may be modified from the naturally occurring units. Naturally occurring M and G alginate units exhibit the same general chemical structure irrespective of their source, although, the distribution and proportions of M and G units will differ depending upon the source. Natural source alginates, for example from seaweed or bacteria, can thus be selected to provide side chains with appropriate M and G units for the ultimate use of the polymer. Isolation of alginate chains from natural sources for use as the side chains herein can be conducted by conventional methods. See Biomaterials: Novel Materials from Biological Sources, ed. Byrum, Alginates chapter (ed. Sutherland), p. 309-331 (1991). Alternatively, synthetically prepared alginates having a selected M and G unit proportion and distribution prepared by synthetic routes analogous to those known in the art can be used as the side chains. Further, either natural or synthetic source alginates may be modified to provide M and G units with a modified structure as long as the polymers with modified side chains still provide a gel or highly viscous liquid by interaction of the alginate units with a divalent metal. The M and/or G units may be modified, for example, with polyalkylene oxide units of varied molecular weight such as shown for modification of polysaccharides in Spaltro (U.S. Pat. No. 5,490,978) with other alcohols such as glycols. Modification of the side chains with such groups generally will make the polymer more soluble, which generally will result in a less viscous gel. Such modifying groups can also enhance the stability of the polymer. Further, the polymers can be modified on the side chains to provide alkali resistance, for example, as shown by U.S. Pat. No. 2,536,893.
Useful polysaccharides other than alginates include agarose and microbial polysaccharides such as those listed in Table 1:
The polymeric backbone section, linkages and side chains may be provided in a number of configurations which configuration will be a factor in the controllability of the polymer properties. The configuration of the polymeric backbone section, the number and location of linkage sites and the type and number of side chains will determine the configuration. Examples of useful configurations are shown in FIG. 1 although the invention is not limited to such configurations and further configurations using the three basic structural units can be provided according to the invention. Especially preferred, however, are polymers having the branched configuration. It is noted that the xe2x80x9cside chainsxe2x80x9d of the linear polymers are on the terminal ends of the backbone, but are still considered side chains herein. Further, the side chains may be present between sections of polymer backbone in an alternating block type configuration.
One preferred embodiment is materials wherein the backbone itself is an alginate. The side chains, for example, may be polyguluronate derived from sodium alginate. A particular example involves cross-linking polyguluronate to itself, via a hydrolytically degradable bond, utilizing a bifunctional cross-linking molecule to form a cross-linked polymer. Dendritic polymers and comb polymers, as described below can also be provided as such materials. These structures can provide a highly cross-linked polymer which would rapidly degrade to low molecular weight components and readily be cleared by the body. To achieve this goal, for example, polyaldehyde guluronate is reacted with hydrazine and sodium borohydride to afford polyhydrazino guluronate. The hydrazine groups on this alginate derived polymer are used to incorporate G-block chains via the their hemiacetal termini. This provides materials from naturally derived polysaccharides with hydrolyzable hydrazone linkages, hence, biocompatible and biodegradable. Hydrolysis of the hydrazone linkage in these materials will lead to short chain polysaccharides that can be excreted by the kidney. Further more, reduction of the hydrazone bond by borohydrides can form a chemically stable hydrazine bond that provide non-degradable materials. Thus, both biodegradable and non-degradable biomaterials can be derived from natural polysaccharides. Cells within the polymer are not damaged by the cross-linking reaction, indicating that these materials are useful for cell transplantation, for example.
Dendrimers provide a particularly interesting backbone structure since they exhibit different properties from the corresponding linear polymers due to the difference in molecular shape and structures. Dendritic molecules can be provided as a backbone with handles to branch off a large number of functional groups in a compact region. Since polypeptides are biodegradable and their degradation products (i.e., the amino acids) are non-toxic, certain polypeptides (e.g. polylysines) can be used as dendritic handles. In connection therewith, soluble polymer supports which combine the advantages of both solid phase and solution phase syntheses can be used to prepare the materials. The most typical soluble polymer supports utilized are comprised of poly(ethylene oxide) (PEO). The reasons are the hydrophilic nature of PEO and insolubility in a variety of organic solvents which is desirable for purification purposes.
A further useful backbone structure is comb polymers which contain many side chains extending from a polymer backbone. Poly(vinyl alcohol) (PVA) provides a particularly useful backbone for comb polymers. The alcohol groups of the PVA can be esterified and subjected to the above-discussed carbodiimide linkage chemistry to provide the side chain linkages.
The materials containing a polymer backbone may be prepared utilizing synthetic methods known in the art, some of which are discussed above, for example in the Biomedical Engineering Handbook, section 4,; see also Odian, Principles of Polymerization, Chapter 9, 2nd ed., (1970). For example, polymeric backbone starting materials can be used which already contain suitable linkage sites, e.g. free amino groups such as certain poly(amino acids), or the polymers can be reacted with linker compounds to provide suitable linkage sites, particularly by the reaction of suitable sites on the polymeric backbone with amino acid derivatives, optionally with the amino groups being protected. Further, some reactive sites on the backbone may be protected to prevent addition of the linker group if it is desired to keep such sites free or to subsequently provide such sites with different linker groups. This chemistry is conventional in the field of linker/polymer formation, especially involving ester, amide, ether and other covalent linkages; see, e.g., Bronzino and Hermanson, cited above. For protective groups, see, e.g., Vogel""s Textbook of Practical Organic Chemistry, 5th ed. p. 550+ and 784+. After removal of the optional protecting groups on the linker, reaction with the side chain of M and/or G units is conducted, preferably through grafting by reductive amination of the reducing end of the side chain with the amino group of the linker, to produce the subject polymers. The side chains are provided as described above from natural sources or synthetically, and may have, optionally, the described modifications they may be bonded as described above, or by other conventional methods.
Another embodiment of synthetic analogues of alginate materials are those provided by covalent crosslinking of the alginate. This covalent crosslinking greatly expands the range of situations in which these materials are useful. One specific application of this modification is the development of matrices of the alginate with shape memory. The crosslinked alginate provides advantageous shape memory properties and compression resistance properties which make them particularly advantageous for use in forming cell transplantation matrices. Shape memory matrices are designed to xe2x80x9crememberxe2x80x9d their original dimensions and, following injection in the body in a compact form (e.g., through a syringe) or other means of placement in the body or in other locations which they may find use, resume their original size and shape. The shape memory property of the alginate is provided by crosslinking thereof. Crosslinking can also improve the compression resistance and/or other mechanical properties of the alginate. Further, a crosslinked alginate can provide a degree of cell adhesion even without use of biologically active cell adhesion ligands. Gelling by divalent cations provides another means of increasing the viscosity and degree of structure of the alginate in addition to the crosslinking. Further, the crosslinked alginate may be covalently bonded to at least one cell adhesion ligand to provide for cell adhesion and maintenance of cell viability.
It is also an object of the invention to provide a process for preparing such crosslinked alginates and to provide compositions and methods utilizing such crosslinked alginates.
The alginate used for crosslinking according to the invention are alginate chains which contain polymerized D-mannuronate (M) and/or L-guluronate (G) monomers, but the term xe2x80x9calginatexe2x80x9d or xe2x80x9calginate chainxe2x80x9d as used herein also is intended to encompass chains wherein such monomers are modified such as described below when they are compatible with the ultimate use and able to be crosslinked covalently. The alginate chain is particularly preferably comprised of oligomeric blocks of M units, G units, M and G units, or mixtures of such blocks. The general structure of an alginate linear copolymer of M and G units is demonstrated by the following general formula: 
The molecular weight of the alginate chain and, thus, the number of units and length of the chains may be selected dependent upon the desired properties of the polymer. In general, the molecular weight of each chain may range from about 1,000 to one million, for example. Higher molecular weight chains, e.g., above about 100,000, are generally useful when more stable alginate polymers are desired and lower molecular weight chains, e.g., below about 30,000 to 50,000, are generally useful when biodegradable species capable of removal through the kidneys or through other normal metabolic functions are desired.
The distribution of M and G units also provides a controllability feature of the invention with a higher ratio of G units generally providing a stiffer alginate material which will hold its shape better. An alginate chain having a percentage of G units based on the total of M and G units of from 10 to 100% is particularly preferred. Increasing or decreasing the number of G units in the chain will also allow for increasing or decreasing, respectively, the rate of gelation of the alginate. Such may be of interest when the alginate is used in an injectable solution and the rate is controlled so that the solution will gel at the appropriate time after injection.
The alginate chain or individual M and/or G units may also be modified from the naturally occurring units. Sources for the naturally occurring alginates and for modified alginates are described above in relation to the alginate side chains for the polymeric backbone embodiment described above.
Furthermore, useful as the alginate starting material are materials having a polymeric backbone to which is linked alginate side chains, as described above. The crosslinking may occur between side chains of the same backbone and/or between side chains of other backbones. It is also possible to have different types of alginate-containing materials with crosslinking provided between alginate sections or chains thereof. Mixtures of any of the above alginate starting materials may also be used.
The crosslinking of the alginates is by action of a crosslinking agent to provide covalent bonding, through the crosslinking agent, from the carboxylic acid groups of the uronic acid of one alginate unit to the carboxylic acid group of the uronic acid of another alginate unit. Such crosslinking is preferably between alginate units from different alginate chains. However, crosslinking may also occur between alginate units of the same chain or, in the case where the alginates are side chains on a polymer backbone as described above, crosslinking may occur between different side chains on the same or differing polymer backbones.
The crosslinking agent may be any suitable agent with at least two functional groups which are capable of covalently bonding to the carboxylic acid groups and/or alcohol groups of the alginate or modified groups therefrom. Crosslinking agents of higher functionality may also be used. For example, polyamines such as bifunctional, trifunctional, star polymers or dendritic amines are useful and these can be made, for example, by conversion from corresponding polyols. Preferred crosslinking agents are those with at least two nitrogen-based functional groups such as, for example, diamine or dihydrazide compounds; non-limiting examples thereof being diamino alkanes, Jeffamine series compounds, adipic acid dihydrazide and putrescine. Particularly preferred as a crosslinking agent is lysine, especially an ester thereof, particularly the methyl or ethyl ester.
The crosslinking agent may also be selected to provide a more or less biodegradable or non-biodegradable bond such that the lifetime of the resulting crosslinked alginate material in its environment, e.g. in vivo, can be modified for the intended utility.
The amide bonds formed when crosslinking with an amine crosslinking agent of alginates are less susceptible to hydrolytic cleavage compared to the acetal linkages between the consecutive uronic acids units of alginates. Therefore, products crosslinked with regular diamines are of relatively low biodegradability in this series of materials, since the polysaccharide (alginate) will degrade before the linking molecules will. To improve upon the rate of biodegradation, a more labile functional group may be incorporated into the crosslinker. Bifunctional biodegradable crosslinkers may be synthesized according to well established chemical pathways. See the following schematic exemplifying preparation of a crosslinking agent with biodegradable ester linking: 
Modification of ethylene glycol to form biodegradable bifunctional crosslinkers. For example, ethylene glycol could be coupled with two N-(t-Boc)glycine using carbodiimide chemistry to yield 1,2-ethylene-(N,Nxe2x80x2-di-t-Boc)glycine intermediate. This intermediate could be deprotected using trifluoroacetic acid in methylene chloride at various temperatures to yield 1,2-ethyleneglycoldiglycinate intermediate. This intermediate could be deprotected using trifluoroacetic acid in methylene chloride at various temperatures to yield 1,2-ethyleneglycoldiglycinate intermediate. In addition to ethylene glycol, other molecules with two terminal alcohol functional groups could be utilized. Moreover, polyols including, e.g., (star shaped or dendritic) could be transformed into similar types of crosslinkers with biodegradable ester functional groups incorporated using parallel chemical pathways.
Preferably, though not necessarily, the crosslinking is facilitated by an activator compound which reacts with the carboxylic acid group of the alginate unit to make it more reactive to the crosslinking agent. Useful activators for making a carboxylic acid group more reactive to the crosslinking agent, particularly an amine functional group of the crosslinking agent, are known in the art. Examples thereof include, but are not limited to, carbodiimides, particularly water-soluble carbodiimides such as, for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC), and CDI (carbodiimidazole).
Also preferred, when using an activator compound, is the use of a stabilizer for stabilizing the resulting activated group. Again, useful stabilizers for the activator groups are known in the art. For carbodiimides, particularly EDC, a useful stabilizer is 1-hydroxybenzotriazole (HOBT) which stabilizes the activated group against hydrolysis. Other useful stabilizers include N-hydroxysuccinimide and N-hydroxysulfylsuccinimide (sulfo-NHS).
The reaction sequence using a lysine ethyl ester crosslinking agent with EDC activator and HOBT stabilizer is shown in the following schematic: 
Reaction pathway of alginate crosslinking. EDC activate carboxylic acid to yield O-acylisourea intermediate. This intermediate reacts with HOBT to form HOBT activated intermediate. Primary amino groups in lysine ethyl ester then couple the activated carboxyl groups of adjacent alginate molecules to form crosslinked alginates.
The crosslinking can generally be conducted at room temperature and neutral pH conditions, however, the conditions may be varied to optimize the particular application and crosslinking chemistry utilized. For crosslinking using the EDC chemistry, optionally with HOBT or sulfo-NHS intermediate steps, pH of from 4.0 to 8.0 and temperatures from 0xc2x0 C. to room temperature (25xc2x0 C.) are optimal and preferred. It is known that higher temperatures are unpreferred for this chemistry due to decomposition of EDC. Similarly, basic pH (e.g., 8-14) is also unpreferred for this reason when using this chemistry.
Other crosslinking chemistries can also be used. For example, using poly(ethylene glycol) (PEG) as a spacer in a crosslinking agent with an N-protected amino acid (see Example 12). Also, crosslinking of oxidized alginate can be conducted with adipic acid dihydrazide. The oxidation results in polyaldehyde alginates (limit oxidized alginates) for crosslinking (See Example 17). Additionally, crosslinking can be effected by light activation using photoreactive materials (See Example 26).
Another method of altering the mechanics of crosslinked systems is by varying the molecular weight between cross-links, Mc, in the polymer network (Peppas and Bar-Howell, Hydrogels in Medicine and Pharmacy. Vol 1, CRC, Boca Raton, pp 28-55, 1986; and, Anseth et al., Biomaterials 17:1647-1657, 1996). Mc may be modified by controlling the extent of cross-linking, or by varying the molecular weight of the cross-linking molecule (Simon et al., Polymer 32:2577-2587, 1991). Both of these strategies may be utilized to alter the mechanical properties of the alginate gels. Covalent cross-linking has been achieved with several different approaches. Cross-linking with lysine results in amide bond formation, which will provide stability and will degrade very slowly. PEG-crosslinkers contain an ester bond, which will be more labile to hydrolysis. Finally, cross-linking of oxidized alginate with adipic acid dihydrazide leads to a hydrazone bond. Importantly, these materials may be both covalently and ionically (e.g., calcium) cross-linked. This may prove advantageous in certain applications in which one desires a two-stage gelling. For example, the polyaldehyde alginates described below will cross-link ionically very quickly (e.g., minutes), while the covalent cross-linking reaction can be designed to occur very slowly (e.g., hours). A surgeon could thus ionically cross-link these polymers to yield a solution which is amenable to injection via a syringe or endoscope, but is viscous enough (viscosity at this stage decreases with increasing extent of oxidation) so that it does not extravasate after being placed. The covalent cross-linking would subsequently harden the implanted material into a more rigid, non-flowable mass.
Crosslinking of the alginate provides a more structured material, the extent of structuring being dependent, at least in part, on the extent of crosslinking. The extent of structuring of the alginate material will also depend, among other factors, upon the extent of gelling through action of the ionic bonding of the divalent metal cation, as discussed above, and upon the nature of the starting alginate material, which as discussed above may be varied, for example, to affect stiffness of the material. Depending on the extent of crosslinking and these other factors, the crosslinked alginate material may run the spectrum through the following forms: a viscous liquid, a swellable gel, a non-swellable gel, a swollen polymer network or a solid matrix, for example.
The extent of crosslinking is a function of the amount of crosslinking agent and crosslinking method used, i.e., the molar percent of crosslinking agent per mole of crosslinkable alginate carboxylic acid groups. The alginate will be increased in viscosity as it is crosslinked. Thus, the extent of crosslinking will be dependent upon the ultimate use. For example, to provide gel materials which have super absorbency properties, it is useful to have a low crosslinking extent, for example, of about 1 to 20%, preferably 1-10%, of crosslinkable groups crosslinked. For tissue matrix materials, for example, the extent of crosslinking is preferably from about 5% to 75%. In a particular embodiment described in the following examples, the alginate is a viscous liquid when the crosslinking agent amount is about 25 mol % or less, a swellable gel when the amount is about 50% and a solid structure which maintains its size and shape when the amount is about 75% or higher. However, the crosslinking chemistry can be selected and optimized to control viscosity even at lower crosslinking extent. In another embodiment, the crosslinking agent is used in a molar amount about equal (i.e., 100 mol %) to the number of crosslinkable alginate carboxylic acid (uronic acid) groups.
Additionally, the crosslinking can be conducted either before, after or simultaneously with the gelling by action of the divalent metal cations. It is preferred for certain applications that the crosslinking be conducted either before or simultaneously with the gelling by divalent cation so as to prevent problems with diffusion of the crosslinking agent to interior portions of the gelled material.
This material may make an ideal two-stage gelling matrix. The extent of oxidation of the alginate in the first step of the synthesis controls the binding sites available for ionic gelling, and thus regulates the viscosity of calcium cross-linked gels. The covalent cross-linking reaction with adipic acid dihydrazide occurs over several hours, and thus can be used to harden the gel slowly. The ultimate mechanical properties of the matrix can be controlled by varying the extent of covalent cross-linking, and this will be a function of the adipic acid concentration. For example, a material largely insensitive to the time of ionic cross-linking time, and with a time frame for ionic cross-linking considerably shorter than that for covalent cross-linking can be designed.
In a further embodiment, the crosslinked alginate is not gelled by action of divalent metal cations at all or is gelled by cations present in vivo only after the delivery of the crosslinked alginate into the biological system, e.g., body.
For the reasons discussed above, the extent of stiffness and matrix structure of the crosslinked alginate materials will be influenced both by the gelling by divalent cation and by the extent and nature of crosslinking. The ability to vary these and other factors provides great flexibility in designing a material which is particularly suited for its ultimate application.
In addition to the type of cell adhesion discussed below, the matrix structure provided by the crosslinked alginates themselves can facilitate cell adhesion type properties, for example, due to trapping of cells in the matrix or action of a crosslinking agent, such as lysine. For example, the crosslinked alginate as a matrix can be introduced for tissue engineering and the cell can migrate into the pores of the matrix in vivo. It is also advantageous, however, to provide the crosslinked alginates with biologically active molecules to facilitate cell adhesion or other biological interaction, as discussed below. The ligands may be added before, during or after crosslinking of the alginate and/or gelling by divalent cations.
To address the relative biological inertness of the synthetically modified polysaccharide or alginate materials discussed above, the polymers can be modified with biologically active molecules. Another aspect of the invention lies in modifying not only the above-discussed synthetic alginate analogues but also the base naturally occurring, modified or analogous alginate materials which are described herein. Even if the alginate or modified alginate material is not provided on a polymeric backbone and/or not crosslinked, the coupling of the alginate with certain biologically active molecules makes it very useful for tissue engineering and other applications.
The polymeric backbone-containing and/or crosslinked alginates and the naturally occurring or modified base alginate materials can be modified with the cell adhesion active molecule(s), for example, by covalent bonding using amide chemistry between the amine groups of the biological molecules and a free carboxylic acid group of the uronic acid residues (of M and G units) of the alginate or other polysaccharide. If the material is crosslinked, bonded to a polymeric backbone and/or otherwise modified, free acid groups must remain to add cell adhesion groups. If the cell adhesion groups are added first, active groups for any subsequent crosslinking, polymer bonding or other modification must remain. Other chemistries can also be used to effect such bonding to the biologically active molecule. For example, alginate or analogous materials can be modified to provide aldehyde groups thereon, which are reactive with the amino terminal of peptides to provide an imine bond which is reduced to a stable amine bond. An example of this chemistry is described in Example 24 herein.
Examples of suitable cell adhesion molecules include known cell attachment peptides, proteoglycan attachment peptide sequences (see Table 2), biologically active proteoglycans (e.g. laminin and fibronectin) and other polysaccharides (e.g., hyaluronic acid and chondroitin-6-sulfate). Examples of other suitable biological molecules include peptide growth factors (such as EGF, VEGF, b-FGF, acidic FGF, platelet-derived growth factor, TGF or TGF-xcex2), and enzymes (Dominguez et al., 1988: Carbodiimide coupling of xcex2-galactosidase from Aspergillus oryzae to alginate. Enzyme Microb. Technol., 10:606-610; and Lee et al, 1993: Covalent Immobilization of Aminoacylase to Alginate for L-h phenylalanine production. J. Chem. Tech. Biotechnol, 58:65-70). Examples of these molecules and their function are shown in the following Table 1.
Particularly preferred as the cell adhesion molecule bonded to the alginate chain are synthetic peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD) which is known as a cell attachment ligand and found in various natural extracellular matrix molecules. Further of interest is GREDVY (endothelial cell specific) peptide. The alginates with such a modification provide cell adhesion properties to the alginate analogue, natural alginate or modified alginate, particularly when used as a cell transplantation matrix, and sustains long-term survival of mammalian cell systems, as well as controlling cell growth and differentiation.
Coupling of the cell adhesion molecules to the alginate can be conducted utilizing synthetic methods which are in general known to one of ordinary skill in the art. A particularly useful method is by formation of an amide bond between the carboxylic acid groups on the alginate chain and amine groups on the cell adhesion molecule. Other useful bonding chemistries include those discussed in Hermanson, Bioconjugate Techniques, p. 152-185 (1996), particularly by use of carbodiimide couplers, DCC and DIC (Woodward""s Reagent K). Since many of the cell adhesion molecules are peptides, they contain a terminal amine group for such bonding. The amide bond formation is preferably catalyzed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which is a water soluble enzyme commonly used in peptide synthesis. An example of such chemistry is shown in the following equation. 
Therein, EDC reacts with carboxylate moieties on the alginate backbone creating activated esters which are reactive towards amines. R-NH2 represents any molecule with a free amine (i.e. lysine or any peptide sequence N-terminus). To reduce unfavorable side reactions, EDC may be used in conjunction with N-hydroxysuccinimide, N-hydroxysulfylsuccinimide or HOBT to facilitate amide bonding over competing reactions.
The reaction conditions for this coupling chemistry can be optimized, for example, by variation of the reaction buffer, pH, EDC:uronic acid ratio, to achieve efficiencies of peptide incorporation between 65 and 75%, for example. Preferably, the pH is about 6.5 to 7.5. The ionic concentration providing the buffer (e.g. from NaCl) is preferably about 0.1 to 0.6 molar. The EDC:uronic acid groups molar ratio is preferably from 1:50 to 20:50. When HOBT is used, the preferred molar ratio of EDC:HOBt:uronic acid is about 4:1:4. The density of cell adhesion ligands, a critical regulator of cellular phenotype following adhesion to a biomaterial. (Massia and Hubbell, J. Cell Biol. 114:1089-1100, 1991; Mooney et al., J. Cell Phys. 151:497-505, 1992; and Hansen et al., Mol. Biol. Cell 5:967-975, 1994) can be readily varied over a 5-order of magnitude density range. An example thereof is shown in FIG. 2.
Both surface coupling, as well as bulk coupling of alginate can be readily obtained with this coupling chemistry. Therefore, by manipulation of surface and bulk coupling, materials having one type of molecule coupled internally in the matrix and another type of molecule coupled on the surface can be provided, for example.
Other methods conventionally known for attachment or immobilization of adhesion ligands may be used, such as discussed in Bronzino cited above, p. 1583-1596.
The biological molecules useful for attachment to the above-described alginate materials are not, however, limited to those providing cell adhesion function. For example, the polymer could be bound to a molecule with antiseptic function when used as a wound dressing, or which provides adhesion tissue specific gene expression, growth factors to enhance proliferation of cells in the environment or vascularation of the tissue or anti-inflammatory activity.
The combination of the alginate and alginate analogue materials with cell adhesion ligands bonded thereto provides a unique three dimensional environment in which the cells interact through various forces for adhesion to the alginate which has many uses, particularly for tissue engineering applications. The cell adhesion ligands provide specific cell membrane receptor sites for the desired cells. The number, type and location of the cell adhesion ligands on the alginate or alginate analogue material will affect the cell adhesion and cell viability maintenance properties and such factors can be varied to suit the particular application. Such applications include tissue engineering methods applied to humans and animals. Preferably, 10xe2x88x9212 to 10xe2x88x924 moles of adhesion molecules per milliliter of hydrated alginate are used; see Massia et al., J. Cell. Biol; Vol. 114, p. 1089-1100 (1991). Also, combinations of the cell adhesion ligands with differing cell adhesion ligands or other bioactive molecules may be utilized according to the invention. Such additional groups may be bonded at other sites on the alginate or to suitable sites on ligands already present on the alginate or alginate analogue material.
The alginate having a polymeric backbone and/or being crosslinked or the natural or modified alginate or other polysaccharide, optionally with bioactive molecules, can create a synthetic extracellular envirornent for mammalian cells that is capable of performing the diverse functions of the natural extracellular matrix (ECM). The materials described herein will, thus, have application in the field of tissue engineering, biomaterials, and in the basic cell biological sciences for studying three dimensional cell interactions and tissue morphogenesis. The materials described herein are advantageous as a model system for creating a synthetic ECM capable of guiding cellular gene expression during in vitro or in vivo tissue formation.
The natural ECM regulates cell growth and differentiation with features that allow the control of the mechanical and chemical environment around the cells (D. E.
Ingber. Mechanochemical Switching between growth and Differentiation ba, Extracellular Matrix, in Principles of Tissue Engineering (Ed, Lanza, Langer and Chick) p. 89-100 (1997)). The alginate and analogue materials are capable of displaying a wide range of mechanical properties and, with covalent modification by the bioactive molecules as described, can display a wide range of biochemical properties, such as connecting mammnalians cell with the extracellular environment which previous cell encapsulation matrices have not been capable of. The covalent modification with bioactive sequences allows the creation of a two-dimensional or three dimensional synthetic extracellular environment capable of providing biochemical signaling in the form of sequestered growth factors, hormones or active sequences within these specific chemicals, and more importantly it will allow mammalian cells to communicate with other cells directly through the alginate material via cell attachment peptides (e.g., RGD, YIGSR, REDV) covalently attached directly to the material. By then controlling the mechanical properties of the alginate materialxe2x80x94for example by the nature of the polymer backbone and/or by crosslinking and/or by modifications of the alginate chain thereof in the manners discussed abovexe2x80x94it will be possible to control the intercellular signaling between the cells and among cell populations (see D. E. Ingber, Mechanochemical Switching between Growth and Differentiation by Extracellular Matrix, in Principles of Tissue Engineering (Ed. Lanza, Langer and Chick )p. 89-100 (1997) and G F Oster, J D Murray, and A K Harris, Mechanical aspects of Mesenchymal Morphogenesis, Journal of Embryology and Experimental Morphology, Vol. 78, p. 83-125 (1983)).
Unmodified alginate has been used as a cell immobilization material for many years due to the stable hydrogels formed with mild gelling conditions. However, the alginate acts only as a neutral agent suspending cells or cell aggregates in three dimensions. By modifying this polysaccharide structurally in the manners discussed above and optionally with cell attachment peptides, growth factors, hormones or ECM binding sequences, for example, the alginate can be transformed into a dynamic, interactive matrix capable of guiding cellular gene expression in space and time. The ability to control the viscoelastic properties of the alginate is an integral aspect in guiding cellular gene expression (see M. Opas, Substratum Mechanics and Cell Differentiation. International Review of Cytology, Vol. 150, p. 119-137 (1994); and G F Oster, J D Murry, and A K Harris, Mechanical aspects of Mesenchymal Morphogenesis, Journal of Embryology and Experimental Morphology, Vol. 78, p 83-125 (1983)) and can be used in model in vitro cell culture systems and tissue engineering applications.
Matrices play a central role in tissue engineering. Matrices are utilized to deliver cells to desired sites in the body, to define a potential space for the engineered tissue, and to guide the process of tissue development. Direct injection of cell suspension without matrices have been utilized in some cases, but it is difficult to control the placement of transplanted cells. In addition, the majority of mammalian cell types are anchorage dependent and will die if not provided with an adhesion substrate.
Alginate materials in polymerized form and/or crosslinked and/or modified with bioactive molecules, as discussed above, can be advantageously used as matrices to achieve cell delivery with high loading and efficiency to specific sites. The materials according to the invention also provide mechanical support against compressive and tensile forces, thus maintaining the shape and integrity of the scaffold in the aggressive environments of the body. This is particularly the case when the alginate is crosslinked to a higher degree. The scaffold provided by these materials may act as a physical barrier to immune system components of the host, or act as a matrix to conduct tissue regeneration, depending on the design of the scaffold.
The first type of scaffolds, immunoprotective devices, utilize a semipermeable membrane to limit communication between cells in the device and the host. The small pores in these devices, e.g., (d less than 10 xcexcm) allow low molecular weight proteins and molecules to be transported between the implant and the host tissue, but they prevent large proteins (e.g., immunoglobulins) and host cells (e.g., lymphocytes) of the immune system from entering the device and mediating rejection of the transplanted cells. In contrast, open structures with large pore sized, e.g., (d greater than 10 xcexcm) are typically utilized if the new tissue is expected to integrate with the host tissue. The morphology of the matrix can guide the structure of an engineered tissue, including the size, shape and vascularization of the tissue.
As discussed above, the alginate, alginate analogue and modified alginate materials of the invention are useful for cell transplantation matrices. These materials can be used to provide such a matrix in any of several ways. For instance, when the matrix is desired to be a temporary matrix for replacement by natural tissue, the material can be designed for biodegradability and system release, for example, by providing hydrolyzable linkages, using relatively low molecular weight alginate chains, biodegradable crosslinking agents, biodegradeable polymer backbones and/or low molecular weight polymer backbone sections. Alternatively, when less degradable matrices are desired, non-hydrolyzable linkages, alginate chains of higher molecular weight, non-degradable crosslinking agents and/or higher molecular weight polymer backbone sections can be used. The many ways in which the properties of the materials can be altered provides a high degree of controllability in providing materials which meet the requirements for the specific application.
In a less degradable form, the matrices can be introduced to the body without cells, but cells will migrate into the matrix, in vivo, and regenerate therein. The alginate or analogue material can be provided in an injectable form, optionally bound to appropriate viable cells, after injection in which case endogenous divalent metal cation in the physiological system after injection causes gelation of the alginate portions of the material. Alternatively, divalent metal cations are added to the solution, for example as a calcium sulfate solution, just prior to injection. As discussed above, the material can be designed to control its rate of gelation to match the ultimate utility. Such injectable solutions can be utilized for delivery of cells to regenerate urologic tissues, for reconstructive surgery, skin replacement, other orthopedic applications or other tissue replacement or repair applications. The alginate-containing materials provide a highly structured, gelled or highly viscous matrix in which the cells are compatible and grow to achieve their intended function, such as tissue replacement, eventually replacing the matrix.
As such, the materials, particularly the polymeric type, may act as analogs to natural glycosamine-glycans and proteoglycans of the extracellular matrix in the body. Furthermore, they can be used to provide preformed gelled or highly viscous matrices bound to cells which may then be surgically implanted into a body. It is of particularly surprising advantage that the materials can be used to implant a matrix which does not contain cells and subsequently the cells can be seeded into the matrix in vivo. The materials optionally may be provided, for example, as a gel, as a viscous solution, as a relatively rigid body, as preformed hydrogel, within a semi-permeable membrane, within microcapsules, etc., and the polymer properties controlled as discussed above to facilitate such applications. The utility of the polymers for cell transplantation and tissue engineering is a significant advance in the art, particularly since it was previously considered not to be practical or possible to achieve such results with synthetic materials; see C. Ezzell, The Journal of NIH Research, July 1995, Vol. 7, p. 49-53.
The materials are also advantageously useful as vehicles for drug delivery particularly for sustained release. For drug delivery application, it is useful that the bioactive molecule, i.e., the drug, be linked to the alginate polymer and/or analogue material by a degradeable bond chosen for controllable release in the system. Other utilities of the materials which may or may not employ a bound biological molecule include, for example, wound dressings, wound healing matrix materials, matrices for in vitro cell culture studies, replacements for conventional industrial alginates used, for instance, as food thickening agents and as printing additives, for example to thicken inks, and similar uses wherein the above-described properties are desired. One particularly advantageous use of the crosslinked materials, not necessarily containing bioactive components, is as highly absorbent materials. Particularly, materials with a low extent of crosslinking, e.g., about 1-20% crosslinking, have this utility. The absorbency property makes them useful, for example, in disposable diaper applications. The controllability of the properties of the synthetic polysaccharides according to the invention and the consistent reproduceability of such selected synthetic polysaccharides makes them particularly advantageous for many applications.
The entire disclosure of all applications, patents and publications, cited above and below, is hereby incorporated by reference.